It is proposed that binding selectivity of Pc responsive antifolates is a result of specific enzyme interactions with bound inhibitors. The applicants will test this hypothesis by comparison of the three dimensional crystal structures of both human and Pc dihydrofolate reductase complexed with the cofactor NADPH and Pc selective antifolates such as PTX, TMQ and TMP, to examine whether observed structural changes are correlated to inhibitor selectivity. Analysis of these data will provide molecular level details of inhibitor-enzyme geometry, hydrogen bonding, conformation and the role of specific active site residues, especially the contribution by the substitution of F31I and N64F between human and PcDHFR, in modulating Pc selectivity. X-ray crystallographic techniques using molecular replacement methods will be used to solve these crystal structures. The rationale for this proposed research is that differences in the structural requirements for antifolate binding to human and Pneumocystis carinii DHFR can be exploited for structure-based drug design of new antifolates with potential as AIDS adjuvants. Since selectivity apparently requires only small changes in enzyme-inhibitor geometry, the investigators propose to look for subtle differences in a series of carefully determined crystal structures of DHFR complexes with Pc selective antifolates. Thus knowledge of the three dimensional structure of enzyme-inhibitor complexes are required to define the mechanism of Pc selectivity and action. The rational structure-based design of new Pc selective antifolates will be carried out in collaboration with Dr. Aleem Gangjee, Duquesne University, who will synthesize new antifolates and Dr. Sherry Queener, Indiana University, who will measure their Pc activity.